Expression, purification, and characterization of a membrane-bound d-amino acid dehydrogenase from Proteus mirabilis JN458

Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.

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Characterization of dye-linked d-amino acid dehydrogenase from Sulfurisphaera tokodaii expressed using an archaeal recombinant protein expression system

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2020.04.008 ◽

2020 ◽

Vol 130 (3) ◽

pp. 247-252

Author(s):

Takenori Satomura ◽

Shin Emoto ◽

Norio Kurosawa ◽

Toshihisa Ohshima ◽

Haruhiko Sakuraba ◽

...

Keyword(s):

Amino Acid ◽

Recombinant Protein ◽

Protein Expression ◽

Recombinant Protein Expression ◽

Expression System ◽

Acid Dehydrogenase ◽

Amino Acid Dehydrogenase ◽

Protein Expression System

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Purification and Characterization of a Membrane-bound Deglycating Enzyme (1-Deoxyfructosyl Alkyl Amino Acid Oxidase, EC 1.5.3) from aPseudomonassp. Soil Strain

Journal of Biological Chemistry ◽

10.1074/jbc.271.51.32803 ◽

1996 ◽

Vol 271 (51) ◽

pp. 32803-32809 ◽

Cited By ~ 23

Author(s):

Amit K. Saxena ◽

Poonam Saxena ◽

Vincent M. Monnier

Keyword(s):

Amino Acid ◽

Acid Oxidase ◽

Purification And Characterization ◽

Amino Acid Oxidase ◽

Membrane Bound ◽

Soil Strain

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Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

10.21203/rs.3.rs-860736/v1 ◽

2021 ◽

Author(s):

Toshihisa Ohshima ◽

Taketo Ohmori ◽

Masaki Tanaka

Keyword(s):

Amino Acid ◽

Colorimetric Assay ◽

Maximal Activity ◽

E Coli ◽

Acid Dehydrogenase ◽

Amino Acid Dehydrogenase ◽

Assay Methods ◽

Ph Conditions ◽

Chromatography Step

Abstract Purpose: The primary aim of this study was the purification and characterization of an NADP-dependent L arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) as a novel amino acid dehydrogenase from Pseudomonas veronii. We then applied the enzyme to an L-arginine assay. Methods: An L-ArgDH gene from P. veronii JCM11942 was amplified by PCR using primers based on the N and C-terminal sequences inferred from a putative L-ArgDH gene (PverR02_12350) found in the P. veronii genome. The L-ArgDH activity of the product expressed in Escherichia coli was confirmed, after which the enzyme was purified, characterized, and applied to an L-Arg microassay. Results: The P. veroniiJCM11942 gene was expressed in E. coli, and the gene product exhibited strong NADP dependent L-ArgDH activity. The crude enzyme was unstable but was stabilized by the presence of 10% glycerol under neutral pH conditions. The enzyme was purified to homogeneity through a single Ni-chelate affinity chromatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed L-arginine oxidation in the presence of NADP, with maximal activity at pH 9.5. The apparent Km values for L-arginine and NADP were 2.5 and 0.21 mM, respectively. A simple colorimetric microassay for L-arginine was achieved using the enzyme. Conclusions: The L-ArgDH gene from P. veronii JCM 11942 was successively expressed in E. coli. The product exhibited NADP-dependent L-ArgDH dehydrogenase activity, and the enzyme was purified and characterized as a novel amino acid dehydrogenase. Furthermore, a simple colorimetric assay for L-arginine using L-ArgDH was achieved. Conflict of interest: The authors declare that they have no competing interests.

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Characterization of NADP-dependent L-arginine dehydrogenase as a novel amino acid dehydrogenase and its application to an L-arginine assay

10.21203/rs.3.rs-860736/v2 ◽

2022 ◽

Author(s):

Toshihisa Ohshima ◽

Taketo Ohmori ◽

Masaki Tanaka

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Maximal Activity ◽

Cell Extract ◽

E Coli ◽

Acid Dehydrogenase ◽

Genome Data ◽

Amino Acid Dehydrogenase ◽

Ph Conditions

Abstract L-Arginine dehydrogenase (L-ArgDH, EC 1.4.1.25) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of L-arginine to the oxo analog in the presence of NADP. Although the enzyme activity is detected in the cell extract of Pseudomonas aruginosa , the purification and characterization of the enzyme have not been achieved to date. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP-dependent L-ArgDH activity. The crude enzyme was unstable under neutral pH conditions, but was markedly stabilized by the addition of 10% glycerol. The enzyme was purified to homogeneity through a single Ni-chelate affinity ch romatography step and consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed l-arginine oxidation in the presence of NADP with maximal activity at pH 9.5. The apparent K m values for l-arginine and NADP were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene ( was determined and the amino acid sequence was deduced from the nucleotide sequence. As an application of the enzyme, simple colorimetric　microassay for L-arginine using the enzyme was achieved.

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